Immunoassay techniques first came into wide usage with the development of radioimmunoassay (RIA) in which the specificity of antigen-antibody binding was coupled with the high sensitivity of nuclear particle detection to detect and quantify antibody-antigen binding in the presence of a large background of non-specific material. Later, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) immunoassays coupled the specificity of antigen-antibody binding with the sensitivity of enzyme chemical reactions to detect and quantify an antigen-antibody binding by producing colored, fluorescent, bio- or chemiluminescent chromophore. EIA and ELISA exhibited an amplification factor as high as 108, allowing sensitivities competitive with RIA without the disadvantages of radioactivity.
Typical ELISA diagnostics relied on an antigen having at least one epitope to which an enzyme-linked antibody could bind with a high affinity. An antigen was affinity-isolated from its biological system and allowed to interact with the enzyme-linked antibody. The enzyme of choice was generally alkaline phosphatase or horseradish peroxidase, both of which generated a colored product upon digestion of appropriate substrates. Although detection of attomole levels of an enzyme has been demonstrated, so that it was, in principle, possible to detect attomole levels of an antigen, traditional immunoassays did not operate at that level of detection because all were limited by non-specific binding of the enzyme-linked antibody to surfaces in the reaction well or vial. This produced a background response which restricted the detection limit of the technique which could not be discriminated against because detection was indirect.
A further limitation of the traditional immunoassays employing optical detection was caused by the limited number of clearly resolvable colored enzyme products, at most two or three, which limited the possibility for an immunoassay to screen for multiple antigens in a single sample. Multiple antigen immunoassays usually focused on a number of separate immunoassays in an array of well plates each requiring its own sample which clearly reduced the utility of this approach. The ideal multi-antigen immunoassay would detect a large number of discrete antigens with high specificity in a single specimen, would cover a large dynamic range, be quantifiable over that range, and could be performed rapidly, that is, in minutes rather than hours, for critical clinical situations and high general throughput.
The sensitivity of EIA and ELISA relied on the specificity of the affinants used to bind with the antigen or antibody being detected. Expensive and hard to produce monoclonal antibodies were usually the reagent of choice because the specificity of monoclonal antibodies is very high. Polyclonal antibodies whose specificity is low could be used in theory but were not a practical choice for a reagent because polyclonal antibodies bind with several species of antigens making the detection of the resulting antibody-antigen complex less specific for a single given antigen species.
Yet another restriction to EIA and ELISA is that they required the antigen-antibody binding to reach an equilibrium for quantification, making the immunoassay take several hours to perform.
Until about 1988, mass spectrometry of proteins and peptides was thought difficult or impossible. At that time Karas and Hillenkamp (Analytical Chemistry, vol. 60, pp. 2299-2301, 1988) demonstrated that proteins could be ejected into the gas phase by embedding them into an organic matrix which was then literally exploded using a pulsed laser beam. This technique is commonly referred to as matrix-assisted laser desorption/ionization (MALDI). When MALDI was coupled to a time-of-flight (TOF) mass spectrometer, a new field of biological mass spectrometry was opened.
While the new MALDI techniques opened the field of biomolecular mass spectrometry, the mass spectrometric analysis of complex biological materials was not possible because of matrix overloading. Recently, Hutchens et al. (Hutchens, T. W. and Yip, T., Rapid Communications in Mass Spectrometry, vol 7, 1993, pp. 576-580.) demonstrated the utilization of affinity capture methods to quasi-purify proteins in a specimen prior to MALDI mass spectrometry. By quasi-purifying the specimen being assayed Hutchens et al. effectively overcame the primary limitation of MALDI mass spectrometry, namely, the suppression of ion signal due to overloading of the matrix. They named their technique “surface-enhanced affinity capture mass spectrometry (SEAC)”. They further demonstrated their technique by using single stranded DNA which they immobilized on the mass spectrometer probe tip to quasi-isolate the protein lactoferrin from preterm infant urine.
More recently Hutchens, T. W. and Yip, T., in an international patent application which was published 8 Dec. 1994 (WO 94/28418), described a method and apparatus for using affinity capture to improve mass spectrometric characterization of biomolecules.
Presently, there is no mass spectrometric immunoassay which is capable of qualitatively and quantitatively determining the presence of single or multiple antigen or antibody species in a specimen. It is toward the fulfillment of that need that the present invention is directed.